To investigate the effect of EGF and/or HA on embryo freezability, three biological replicates of the blastocyst stage embryos cultured in BM (n = 67), BM + EGF (n = 59), BM + HA (n = 120) and BM + EGF + HA (n = 63) were subjected to the cryotolerance test. Briefly, the day 7 blastocysts were washed with D-PBS containing 5% PVA and then transferred to a solution consisting of 1.5 M ethylene glycol and 0.1 M sucrose (BoviFreeze, Minitube GmbH, Tiefenbach, Germany). Groups of 10–15 blastocysts were loaded into an open-pulled straw (Minitube GmbH, Tiefenbach, Germany) and then immediately plunged into the programmable freezer “Freeze Control” (Consarctic, Germany). Seeding was accomplished manually with forceps cooled in liquid nitrogen at a temperature of -6°C, followed by a cooling to -30°C at a rate -0.3°C /min. Thereafter, the straws were kept in liquid nitrogen. One week later, the embryos were thawed and washed with SOF media supplemented with fatty acid free BSA. Finally, the embryos were cultured in 400 μl culture media in four well dishes (Nunc, Roskilde, Denmark) covered with mineral oil at 38.7°C in 5% CO2 and humidified air. The expansion and hatching rates were determined at 24, 32, 48 and 56 hr post thawing.
Four well dishes
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, Japan
Four-well dishes are laboratory equipment designed for cell culture applications. They provide a multi-compartment format to facilitate parallel experiments or observations. The dishes offer a standardized size and configuration to support various scientific workflows.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fgf10, by R&D Systems (1 mentions)
Matrigel, by R&D Systems (1 mentions)
Matrigel, by Corning (1 mentions)
Tcm 199, by Thermo Fisher Scientific (1 mentions)
17b estradiol, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Strontium chloride, by Merck Group (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Axiocam 208 camera, by Zeiss (1 mentions)
Zen 3, by Zeiss (1 mentions)
Fulvestrant, by Selleck Chemicals (1 mentions)
G mops plus, by Vitrolife (1 mentions)
G 1 plus, by Vitrolife (1 mentions)
G2 plus, by Vitrolife (1 mentions)
Inverted microscope, by Nikon (1 mentions)
M199 medium, by Thermo Fisher Scientific (1 mentions)
Gestron 1500, by Kyoritsu Seiyaku (1 mentions)
23 protocols using four well dishes
1
Evaluating Embryo Cryotolerance with EGF and HA
2
Embryonic Stomach Epithelial Cell Culture
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Epithelial cells were gathered from embryonic stomachs by FACS as described above. Four-well dishes (Nunc) were coated with 4% Matrigel (Corning) in 375 µL RPMI for 60 min, and then sorted cells were replated in Advanced DMEM containing B27, N2, HEPES, Glutamax, P/S, and 2.5% Matrigel with or without Fgf10 (R&D Systems). Medium was changed every 2 days.
3
Bovine Oocyte In Vitro Maturation
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Bovine ovaries were obtained from a local abattoir. COCs were collected by aspiration from follicles 2–8 mm in diameter.
Collected COCs were washed three times in PBS supplemented with 3 mg/ml BSA (crystallized BSA, Sigma-Aldrich, St. Louis, MO, USA),
100 IU/ml penicilline G potassium (Meiji Seika, Tokyo, Japan) and 100 μg/ml streptomycin (Meiji Seika). The COCs were cultured in
groups of 50 in four-well dishes (Nunc, Roskilde, Denmark) with 500 μl TCM-199 (Gibco-BRL, Grand Island, NY, USA) supplemented
with 1, 5, 10 and 15% sericin solution (substantially 0.01, 0.05, 0.1 and 0.15% sericin/medium) or 5% FBS (Tissue Culture
Biologicals, Tulare, CA, USA), 1 μg/ml estradiol (17b-estradiol, Sigma) and 0.002 AU/ml FSH (Antrin R, Kawasakimitaka, Tokyo,
Japan) for 20–22 h at 38.5 C in an atmosphere of 5% CO2 in air with maximum humidity. After IVM, the nuclei of oocytes
were stained with 1% orcein, and the metaphase II (MII) rates were examined.
Collected COCs were washed three times in PBS supplemented with 3 mg/ml BSA (crystallized BSA, Sigma-Aldrich, St. Louis, MO, USA),
100 IU/ml penicilline G potassium (Meiji Seika, Tokyo, Japan) and 100 μg/ml streptomycin (Meiji Seika). The COCs were cultured in
groups of 50 in four-well dishes (Nunc, Roskilde, Denmark) with 500 μl TCM-199 (Gibco-BRL, Grand Island, NY, USA) supplemented
with 1, 5, 10 and 15% sericin solution (substantially 0.01, 0.05, 0.1 and 0.15% sericin/medium) or 5% FBS (Tissue Culture
Biologicals, Tulare, CA, USA), 1 μg/ml estradiol (17b-estradiol, Sigma) and 0.002 AU/ml FSH (Antrin R, Kawasakimitaka, Tokyo,
Japan) for 20–22 h at 38.5 C in an atmosphere of 5% CO2 in air with maximum humidity. After IVM, the nuclei of oocytes
were stained with 1% orcein, and the metaphase II (MII) rates were examined.
4
Diploid Parthenogenetic Oocyte Development
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The oocytes were induced for diploid parthenogenetic development by 6 h incubation in potassium simplex optimized medium (KSOM; MR-121-D, EmbryoMax) supplemented with 5 mM strontium chloride (439665, Sigma-Aldrich), 2 mM EGTA (E3889, Sigma-Aldrich) and cytochalasin B (C6762, Sigma-Aldrich) in a -incubator with 5% and 37°C, as described by Kishigami and Wakayama.20 (link) Then, the oocytes were washed and cultured in KSOM in a -incubator. The oocytes were observed after 24 and 72 h of incubation to assess the development and calculate two-cell embryos and blastocysts. Laser-treated and control groups were cultured in four-well dishes (Nunc) in separate wells.
5
Porcine Oocyte Isolation and Culture
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Porcine ovaries of prepubertal gilts, 6–8 months old and 90–120 kg in weight, were collected at a local abattoir as a waste material and transported to the laboratory in a thermo-flask at 38 °C. The contents of small (1–2 mm in diameter) and medium-size antral follicles (3–6 mm in diameter) were aspirated with a syringe connected to a 20 G needle, pooled in a test tube, and allowed to sediment for 10 min. The sediment was washed twice with HEPES buffered porcine X medium (PXM-Hepes) [30 (link)], placed in a Petri dish, and the COCs were collected with a pipette. Only COCs surrounded by a compact multi-layered cumulus were selected for experiments. Groups of 50 COCs were cultured for 44 h in four-well dishes (Nunclon, Roskilde, Denmark) in 0.5 ml of culture medium at 38.5 °C in a humidified atmosphere of 5% CO2. The diameter of some COCs and denuded oocytes isolated from small and medium follicles was measured by Axiocam 208 camera using Zen 3.6 software (Zeiss).
6
Bovine Cumulus-Oocyte Maturation Protocol
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Bovine cumulus–oocyte complexes (COCs) were recovered and in vitro matured as previously described [61 (link)]. Briefly, immature COCs were obtained by aspirating follicles (2–8 mm) from the ovaries of heifers collected at the slaughterhouse. International Embryo Transfer Society (IETS) classes I-III COCs were matured for 24 h in groups of approximately 50 COCs per well in four-well dishes (NUNC, Roskilde, Denmark) in 500 μL of in vitro maturation medium “IVM” (TCM 199 (M4530) supplemented with 10% (v/v) fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (E4127). The culture conditions were 38.5°C, 5% CO2 in air and maximum humidity.
7
Bovine Oocyte Maturation and Fertilization
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The COCs were washed three times with tissue culture medium 199 (TCM-199) supplemented with 3% foetal bovine serum (FBS; Medicorp, Montreal, QC, Canada) before being placed in humidified air (5% CO2) at 37°C for 2 h to equilibrate the pH. The maturation medium consisted of TCM-199 plus 10% FBS and 200 mM pyruvate, 50 mg/mL of gentamicin, 100 μg/mL of FSH (Gonal-f; Serono Canada Inc., Ontario, Canada) and different concentrations of E2 (10−6 mM, 10−5 mM, 10−4 mM or 10−3 mM) or 2.9 nM fulvestrant. fulvestrant (Selleckchem, Houston, TX, USA) is an estrogen receptor antagonist. A 400-μL maturation medium was used to grow ~50 COCs in four-well dishes (Nunc, Roskilde, Denmark). The cells were supplemented with 200 μL of oil overlay for 24 h at 37°C in humidified air (5% CO2). Mature COCs were divided into two sets in each treatment group: the first set was used to evaluate cumulus expansion and determine the expression of cumulus-expansion-related factors and OSFs, and the remaining COCs were subjected to in vitro fertilisation (IVF) to assess their subsequent development.
8
Intracytoplasmic Sperm Injection Protocol
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Sperm preparation was proceeded by centrifugation in QUINN’s Sperm Washing Medium (SAGE, USA) at 250 g for 10 min. Before the ICSI procedure, the spermatozoons were immobilized in the drop of 7% PVP (Polyvinylpyrrolidone, SAGE, USA). Intracytoplasmic sperm injection was performed for all patients with a standard micromanipulators (Narishige, Japan) and inverted microscope (Nikon, Japan/Leica, Germany). Spermatozoa was microinjected with a × 200 magnitude in a drop of G-MOPS PLUS (Vitrolife, Sweden). After ICSI, oocytes were allocated into 500 μL of G-1 PLUS culture medium (Vitrolife, Sweden) in four-well dishes (Nunc, USA) and cultured at 37 °C, 6% CO2, 5% O2. In day 3, G-1 PLUS culture medium was replaced with G-2 PLUS (Vitrolife, Sweden).
9
In Vitro Maturation of Oocytes: Ochratoxin A Effects
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In vitro maturation (IVM) was performed as reported previously (Mastrorocco et al. 2019 (link)). IVM medium composition is reported in Online Resource 1 . COCs were placed in four-well dishes (Nunc Intermed, Roskilde, Denmark) containing 400 μL of IVM culture medium perwell of a 4-well dish, covered with paraffin oil and cultured in vitro for 24 h at 38.5 °C under 5% CO2 in air. On the day of experiments, serial log-dilutions of OTA stock solution with IVM medium were made to cover a concentration range from 10 to 0.0001 μmol/L, according to the requirements of the experimental design. The highest concentration was selected on the basis of published data for mice (Huang and Chan 2014 (link)) while the nanomolar concentration was chosen as corresponding to OTA blood levels in sheep, after oral ingestion of OTA-contaminated feed (Blank et al. 2003 ). IVM medium containing 1% MeOH was used as vehicle control. For each set of experiments, at least two runs/replicates were performed, where a run/replicate was a group of 20–25 COCs cultured for IVM in one well of a 4-well Nunc plate.
10
Bovine Oocyte Maturation Protocol
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Bovine ovaries were recovered from a local slaughterhouse, and immature cumulus-oocyte complexes (COCs) were obtained by aspirating follicles (2–8 mm) from the ovaries of mature heifers and cows. After selection, COCs with homogeneous cytoplasm and intact cumulus cells (grade I and II) were maturated in groups of 50 COCs per well in four-well dishes (Nunc, Roskilde, Denmark) containing 500 µL maturation medium (TCM-199), supplemented with 10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF). Oocytes were IVM for 24 h at 38.5 °C, with 5% CO2 in air with maximum humidity.
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